这项工作得到了安大略省癌症研究研究所、加拿大创新基金会、渥太华区域癌症基金会和特里福克斯研究所的资助。K.J.A. 得到了加拿大 Vanier 研究生奖学金、加拿大卫生研究所硕士奖和安大略省研究生奖学金的支持。

<ol>
	<li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+have+RNAi+screens+taught+us+about+viral-host+interactions?.">What have RNAi screens taught us about viral-host interactions?.</a> Curr. Opin. Microbiol. 12 (4), 446-452 (2009).</li><li>Panda, D., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+genomic+screening:+elucidating+virus-host+interactions.">Cell-based genomic screening: elucidating virus-host interactions.</a> Curr. Opin. Virol. 2 (6), 784-792 (2012).</li><li>Houzet, L., Jeang, K. 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C., Haas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-host+interactomes+and+global+models+of+virus-infected+cells.">Virus-host interactomes and global models of virus-infected cells.</a> Trends Microbiol. 19 (10), 501-508 (2011).</li><li>Gao, S., Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+RNA+interference+high-throughput+screening+technology+in+cancer+biology+and+virology.">Applications of RNA interference high-throughput screening technology in cancer biology and virology.</a> Protein Cell. 5 (11), 805-815 (2014).</li><li>Kilcher, S., Mercer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+approaches+to+study+virus+entry+and+infection.">Next generation approaches to study virus entry and infection.</a> Curr. Opin. Virol. 4, 8-14 (2014).</li><li>Ramage, H., Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-Host+Interactions:+From+unbiased+genetic+screens+to+function.">Virus-Host Interactions: From unbiased genetic screens to function.</a> Annu. Rev. Virol. 2 (1), 497-524 (2015).</li><li>Mercer, J., Snijder, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+screening+reveals+proteasome-+and+Cullin3-dependent+stages+in+vaccinia+virus+infection.">RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection.</a> Cell Rep. 2 (4), 1036-1047 (2012).</li><li>Sivan, G., Martin, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+genome-wide+RNAi+screen+reveals+a+role+for+nuclear+pore+proteins+in+poxvirus+morphogenesis.">Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (9), 3519-3524 (2013).</li><li>Beard, P. M., Griffiths, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loss+of+function+analysis+of+host+factors+Influencing+vaccinia+virus+replication+by+RNA+Interference.">A loss of function analysis of host factors Influencing vaccinia virus replication by RNA Interference.</a> PLoS ONE. 9 (6), e98431 (2014).</li><li>Teferi, W. M., Dodd, K., Maranchuk, R., Favis, N., Evans, D. 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作者没有什么可透露的。

在这里, 我们提出了一个使用高通量 rna 干扰筛选的协议, 以确定宿主目标, 可以操作, 以加强溶瘤病毒治疗。这已成功地通过溶瘤马拉巴病毒和溶瘤痘苗病毒进行了测试, 但是, 正如所指出的, 它可以适应其他溶瘤病毒或其他病毒的使用, 通常用于识别调节病毒复制的宿主基因。该筛选协议还旨在确定增加或减少传播的基因, 但它可以很容易地修改其他读数。例如, 我们的马拉巴病毒的屏幕被设计用来...

高通量全基因组 rna 检测已被证明是一个宝贵的工具, 以揭示生物洞察力在不同的研究领域, 包括病毒生物学。在过去的十年左右, 许多团体进行了基于细胞的大规模 rna 干扰筛查, 以广泛识别调节病毒复制的宿主基因 (在1,2,3,4,5,6, 7). 有趣的是, 大量的这些屏幕已经进行了癌症细胞和一些病毒, 目前溶瘤病毒候选包括痘苗病毒 8, 9, 10, 粘液瘤病毒11, 单纯疱疹病毒12, 水泡口炎病毒13,14和马拉巴病毒15。虽然大多数研究的重点是确定影响病毒复制的某些方面的宿主因素, 而不是确定增强溶瘤病毒治疗的 biotherapeutic 目标, 但有价值的数据可以从这些数据集中挖掘出来。这方面。很明显 , 确定可纵以加强溶瘤病毒治疗的宿主目标的强大潜力尚未得到充分利用。
目前, 大量的溶瘤病毒平台在临床前和临床研究中被测试, 其中包括单纯疱疹病毒、呼肠病毒和痘苗病毒, 这些都在后期试验中取得了明显的成功。对于大多数的这些平台, 已经致力于改变病毒基因组, 以加强治疗。例如, 为了增加肿瘤特异性, 特定的病毒基因已被删除或变异, 以显著限制病毒复制在正常细胞, 但不是在肿瘤细胞。在某些情况下, 转基因已被添加如 GM-脑脊液 (粒细胞巨噬素集落刺激因子), 以事实免疫应答或 NIS (碘化钠转运体), 使体内成像和细胞 radioiodide 吸收治疗目的.然而, 很少有工作专注于操纵宿主/肿瘤基因和探索宿主/肿瘤基因对溶瘤病毒复制的影响。随着高通量筛选技术的出现, 现在可以在基因组范围内探测宿主病毒相互作用, 并破译操作宿主基因组以增强溶瘤病毒治疗的机会 (16, 17,18)。
我们报告的第一项研究表明, 使用高通量基因筛选的概念证明, 以确定宿主目标, 可以操纵, 以加强溶瘤病毒治疗。为了发现调节马拉巴病毒裂癌的宿主基因, 该溶瘤 rhabdovirus 目前正在第一阶段和第二期试验中进行测试, 我们在三种不同的肿瘤细胞系中进行了全基因组 rna 检测, 并与 siRNA (短干扰 RNA) 重复。图书馆目标18120基因15。通过对筛查发现的通路进行分析, 揭示了 ER (内质网) 应力响应通路的富集。我们在这些路径中选择了10次命中, 以便使用 siRNA 与主屏幕中不同的目标序列进行二级验证。作为三级验证的一部分, 我们进行了 IRE1α (肌醇要求酶1α) 的抢救实验, 以确认命中目标。体外和体内使用 IRE1α的小分子抑制剂进行测试, 从而显著提高了溶瘤的功效。
Workenhe et 。12还使用一个聚集的慢病毒载体 shRNA (短发夹 RNA) 库对16056个基因进行了全基因组的 rna 干扰筛查, 以确定限制人疱疹病毒1型 (HSV-1) 突变体 KM100-mediated 裂癌的宿主因素癌细胞。在主屏幕上, 他们确定了343个基因的击倒, 导致 KM100-infected 细胞对模拟感染细胞的细胞毒性增强;他们选择了24这些基因进行二次验证。在24种基因中, 有8个基因在二次筛中被证实, 其中之一是 SRSF2 (丝氨酸/精氨酸-富含剪接因子 2), 随后在三级验证中使用基因抢救实验证实。该小组接着确定了一种 DNA 拓扑异构酶抑制剂化疗, 减少了 SRSF2 的磷酸化, 并表明 HSV-1 KM100 与此抑制剂的联合治疗导致了吐蕃肿瘤小鼠的延长生存。
在上述研究中, 遵循了类似的工作流程。每一个都是从一个主要的全基因组的 rna 干扰屏幕开始, 其次是在主屏幕上确定的点击次数中的辅助屏幕。其次是三级验证研究, 其中包括确认命中是在目标通过执行基因抢救实验在体外通过操纵目标基因产品在体内进行最终验证。在本文中, 我们首先提供了一个用于开发和验证高通量 siRNA 筛选试验的通用协议, 它涉及确定最佳转染条件、病毒数量和感染长度。我们还提供了一个详细的协议, 用于进行一个主要的高通量 siRNA 屏幕, 以及对执行次屏幕验证和三级验证的方法的总体描述。

<table><tbody><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>siRNA library</td><td>GE Dharmacon</td><td>G-005005-02</td><td></td></tr><tr><td>Corning 384-well plate</td><td>Corning</td><td>3985</td><td></td></tr><tr><td>Falcon 384-well plate</td><td>Becton Dickinson</td><td>353962</td><td></td></tr><tr><td>Water</td><td>Sigma</td><td>W4502</td><td></td></tr><tr><td>siGenome SMARTpool PLK1</td><td>GE Dharmacon</td><td>M-003290-01</td><td></td></tr><tr><td>AllStars Negative Control siRNA</td><td>Qiagen</td><td>SI03650318</td><td></td></tr><tr><td>siGenome Non-targeting siRNA Pool #2</td><td>GE Dharmacon</td><td>D-001206-14-20</td><td></td></tr><tr><td>DMEM/HIGH Glucose</td><td>GE Healthcare Life Sciences</td><td>SH30022.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F15051-500ML</td><td></td></tr><tr><td>HEPES solution (1M)</td><td>Sigma</td><td>H3535-100ML</td><td></td></tr><tr><td>Penicillin-Streptomycin 100X solution</td><td>GE Healthcare Life Sciences</td><td>SV30010</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo Fisher Scientific</td><td>2500056</td><td></td></tr><tr><td>DPBS/Modified</td><td>GE Healthcare Life Sciences</td><td>SH30028.02</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778100</td><td></td></tr><tr><td>Oligofectamine</td><td>Thermo Fisher Scientific</td><td>1225011</td><td></td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>22600-050</td><td></td></tr><tr><td>Resazurin sodium salt</td><td>Sigma</td><td>R7017-5G</td><td></td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H21492</td><td></td></tr><tr><td>Formaldehyde (37% by weight)</td><td>Fisher Scientific</td><td>F79-4</td><td></td></tr><tr><td>500 ml bottles</td><td>Corning</td><td>430282</td><td></td></tr><tr><td>Adhesive Sealing Film For Microplates</td><td>Excel Scientific</td><td>361006007</td><td></td></tr><tr><td>Peelable Heat Sealing Foil</td><td>Thermo Fisher Scientific</td><td>AB-3720</td><td></td></tr><tr><td>BioTek MicroFlo Select Dispenser cassette</td><td>Fisher Scientific</td><td>11-120-625</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MicroFlo Select (liquid dispenser)</td><td>Fisher Scientific</td><td>11120621</td><td></td></tr><tr><td>BioTek Synergy HT plate reader</td><td>Biotek</td><td>N/A</td><td></td></tr><tr><td>Opera Imaging and Analysis Instrument</td><td>PerkinElmer</td><td>HH10000115</td><td></td></tr><tr><td>KiNEDx Robotic Arm</td><td>Peak Analysis and Automation (formerly Peak Robotics)</td><td>KX-300-660</td><td></td></tr><tr><td>Cytomat 24C Series Automated Incubator</td><td>Thermo Fisher Scientific</td><td>50080227</td><td></td></tr><tr><td>JANUS Automated Workstation</td><td>PerkinElmer</td><td>AJI4M01</td><td></td></tr><tr><td>Plate Carousel</td><td>Peak Analysis and Automation (formerly Peak Robotics)</td><td>N/A</td><td></td></tr><tr><td>Alps 3000 plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-3000</td><td></td></tr></tbody></table>

genome-wide rnai screening to identify host factors that modulate oncolytic virus therapy

高通量全基因组 rna 干扰 (RNA 干涉) 筛选技术已被广泛用于发现影响病毒复制的宿主因素。在这里, 我们提出了这项技术的应用, 以揭露宿主目标, 专门调节复制的马拉巴病毒, 溶瘤 rhabdovirus, 和痘苗病毒的目的是加强治疗。虽然该协议已测试用于溶瘤马拉巴病毒和溶瘤痘苗病毒, 这种方法适用于其他溶瘤病毒, 也可以用来确定主机目标, 调节病毒复制的哺乳动物细胞在秘书长.本议定书介绍了哺乳动物细胞高通量 rna 干扰筛查的研究进展和验证, 主要的考虑因素和准备步骤对进行一次高通量的 rna 干扰屏幕, 以及一步一步指南为进行一次高通量的 rna 干扰屏幕;此外, 它概括地概述了进行二次屏幕验证和三级验证研究的方法。高通量 rna 干扰筛选的好处是, 它允许一个人以一种广泛而无偏见的方式对病毒复制的任何方面进行分类, 这些因素可以开发出一种体外检测方法, 如传染性、爆裂大小、和细胞毒性。它有能力揭露 biotherapeutic 的目标, 根据目前的知识。

在图 1中介绍了用于识别宿主目标以增强溶瘤病毒治疗的工作流的概述。
如图 1所示, 进行高通量干扰屏幕的关键第一步是检测开发。图 2A提供了用于转染优化分析的样板布局。图 2B由预期结果的代表性图像组成,图 2C</stron...

Watch this Scientific Journal Video about Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy at JoVE.com

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

在这里 , 我们描述了一种使用高通量 rna 干扰筛选的协议 , 以发现宿主目标可以作来增强溶瘤病毒治疗 , 特别是 rhabodvirus 和痘苗病毒治疗 , 但它可以很容易地适应其他溶瘤病毒平台或发现宿主基因, 通常调制病毒复制。

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

genome-wide-rnai-screening-to-identify-host-factors-that-modulate

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.0K 次观看.  Children's Hospital of Eastern Ontario (CHEO) Research Institute. 在这里 , 我们描述了一种使用高通量 rna 干扰筛选的协议 , 以发现宿主目标可以作来增强溶瘤病毒治疗 , 特别是 rhabodvirus 和痘苗病毒治疗 , 但它可以很容易地适应其他溶瘤病毒平台或发现宿主基因, 通常调制病毒复制。

视频: Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

基于DNA载体的RNA干扰在癌症研究基因功能

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

医学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

癌症研究

基因组规模筛选，用于识别和比较肿瘤中的治疗靶点

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

作者聚焦:通过基因组规模 shRNA 筛选寻找恶性周围神经鞘瘤的新治疗靶点 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

生物学

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

摘要

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此视频中的章节

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

基于DNA载体的RNA干扰在癌症研究基因功能

高通量筛选RNA病毒的广谱化学抑制剂

合并的shRNA文库筛选，以确定调节耐药性表型的因素

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

小核糖核酸病毒取向的微小RNA基于调控